Probes for hybridization to nucleic acids (NA), with which we refer to both deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), are used to demonstrate the presence of specific target sequences (TS) in complex mixtures. Traditional hybridization methods, as first described by Gillespie and Spiegelman (J. Mol. Biol. 12, 829, 1956), employ a probe based on an oligodeoxyribonucleotide equipped with a reporter group (RG) that usually is a radioisotope, and encompasses usually the following steps: the nucleic acid to be tested is immobilized on a paper, glass bead or plastic surface; an excess of probe complementary to the target sequence is added; the probe is allowed to hybridize; non-hybridized probe is removed; remaining probe bound to the immobilized target sequence is detected.
Non-hybridized probe is removed by extensive washing. This is usually the most time consuming and critical step in the procedure. Since the properties of non-hybridized and hybridized probe are not distinguishable, it is necessary that essentially all non-hybridized probe is removed. Since the hybridized probe is only attached through its interaction with the target sequence also some of it will be removed by washing, as well as some hybrids between TS and probe where TS was not sufficiently immobilized. Further, some probe may stick directly to the surface giving rise to a background signal. Finally, the requirement that non-hybridized probe must be removed makes in vivo and real time detection impossible.
A few methods to demonstrate hybridization without having to remove non-hybridized probe, so called homogeneous probing techniques, have been described.
Bannwarth et al., (Helvetica Chimica Acta, 71, 2085, 1988) have developed a method with probes composed of an oligodeoxyribonucleotide equipped with a ruthenium complex, where hybridization can be demonstrated from measurements of the probe fluorescence lifetime. Although the strategy is elegant, its application is limited to specialized laboratories that have sophisticated instrumentation, and can only be used by people with special training. Further, the ability of the method to distinguish hybridized and non-hybridized probe is not too good, particularly not in biological samples that may contain components that affect the probe fluorescence life time.
Barton J., (U.S. Pat. No. 5,157,032) describes a probe composed of a DNA-chain modified with a metal-ligand complex whose fluorescence intensity increases upon hybridization. These probes obtain only a modest fluorescence upon hybridization (a fluorescence quantum yield of 0.007, has been reported, Jenkins & Barton, J. Am. Chem. Soc., 114, 8736, 1992), which gives low sensitivity. Further, the probes are dicationic (charge +2), which leads to considerable non-specific contribution to the interaction and consequently a decreased ability to distinguish different sequences.
Yamana et al., (Nucl. & Nucl. 11 (2-4), 383, 1992) describe a probe composed of an oligonucleotide modified with pyrene, which under optimal conditions gives a 20-fold increase in fluorescence upon hybridization. The method has several disadvantages. Pyrene has complicated photophysics and its absorption and fluorescence properties depend on its closest surrounding; for example, it has a large tendency to form excimers (J. Michl & Erik W. Thulstrup in Spectroscopy with polarized light, 1.sup.st Ed. VCH, 1986, ISBN 0-89573-346-3). Further, pyrene emits ultraviolet light (below 450 nm) that cannot be seen by the naked eye. Finally, pyrene is toxic (Yoshikawa et al., Vet. Hum. Toxicol. 29, 25, 1987).
Linn et al., (EP 0710 668 A2, U.S. Pat. No. 5,597,696) and Ishiguro et al., (Nucl. Acids Res. 24, 4992, 1996) describes probes composed of an oligonucleotide and an asymmetric cyanine dye. The fluorescence properties, such as fluorescence polarization, fluorescence lifetime and fluorescence intensity, of these probes are changed upon hybridization. These probes have several disadvantages and limitations. Measurements of fluorescence polarization and fluorescence lifetime require sophisticated and expensive instrumentation, and must be performed by people with specialist training. The change in fluorescence intensity is modest (a 4-fold increase under optimal conditions has been reported), making probing very sensitive to background, particularly at conditions that require excess of probe.
Heller et al., (EPA 070685) and Cardullo et al., (Proc. Natl. Acad. Sci. USA, 85, 8790-8794, 1988) describe a probe based on simultaneous hybridization of two DNA-based probes to close-lying sequences. One probe is modified in the 3'-terminus of the DNA chain with a donor fluorophore and the other probe is modified in the 5'-terminus with an acceptor fluorophore. When they are in proximity fluorescence energy is transferred from the donor to the acceptor fluorophore, which can be detected. The fluorophores are far apart in solution, but are brought together when the probes hybridize to TS by binding with the 3'-terminus of one probe next to the 5'-terminus of the other probe. The strategy has several disadvantages. It is necessary to distinguish fluorescence intensity of different wavelengths, since hybridization does not give rise to a significant change in total fluorescence, but only a change in the wavelength of fluorescence. The system is not suitable for quantitative determination of TS, since energy transfer efficiency depends on factors such as the distance between the fluorophores and their relative orientation (Forster, Ann. Phys. (Leipzig) 2:55-75, 1948), which may depend on the probed sequence. The strategy has fundamental problems with background fluorescence, since the light used to excite the donor does also to some degree excite the acceptor leading to a non-specific background signal. Finally, the requirement that two probes bind simultaneously to the target sequence results in slow hybridization kinetics making the technique less suitable for real time detection.
Another technique based on a pair of oligonucleotides was described by Morrison (EPA 87300195.2; U.S. Pat. No. 4,822,733; Analyt. Biochem. 183, 231-244, 1989; Biochem. 32, 3095-3104, 1993). These oligonucleotides are complementary to each other and also to the two strands of the target sequence. Both have a fluorophore in the 3'-terminus and a quencher in the 5'-terminus. When these pair with each other the quenchers at the 5'-terminus are in immediate proximity of the fluorophores at the 3'-terminus quenching their fluorescence. However, if the probe instead binds to TS fluorescence is observed. With this strategy one has two opposing design problems: It is desirable to have a high probe concentration to obtain fast hybridization kinetics, but simultaneously it is desirable to have a low probe concentration to minimize the background luminescence from free probes that have found neither TS nor a complementary probe to bind. Probing is performed by first heating the sample to separate the strands of both the probe molecules and the dsNA, and then the temperature is lowered to allow the probe to hybridize to TS. Unhybridized probe must, however, find a complementary probe to become quenched, until then it give rise to the same signal as probes hybridized to TS. Since probe is usually used in large excess, it make take considerable time before the background has dropped to an acceptable level making the strategy unsuitable for real-time detection. Finally, these probes are only applicable to double stranded TS. Tyagi, S., (PCT-WO 9513399; Nature biotech. 14, 303-307, 1989) describes `molecular beacons` that are based on a probe with two chromophores, one at each end. These are chosen such that one chromophore quenches the fluorescence of the other when they are in proximity. The probe is designed to form secondary structure in solution that brings the two ends of the probe together, resulting in fluorescence quenching. This structural requirement is the first limitation of the probe since it must contain sequences that produces a particular secondary structure. As a consequence the probes are complementary also to other sequences than those they are designed to recognize, i.e., a probe is never unique for single TS. A further disadvantage is that probing is limited to a narrow temperature range, since both the hybrid between probe and TS and the secondary structure in the free probe must be stable. Temperatures at which TS does not hybridize to complementary NA, for example, can not be used. Further, thermal motion, which is significant already at room temperature, decreases the quenching efficiency, making it often necessary to use even lower probing temperatures, which decreases the specificity of the probing reaction.
One objective of the present invention is to overcome the limitations discussed above with traditional methods and also the limitations of the present homogeneous methods.
Further objectives with the present invention are:
that pretreatment of the sample, such as degradation to smaller fragments, should not be necessary, PA1 that target sequences are detected through hybridization with a probe that generates a signal, but which in non-hybridized state generates a much smaller, preferably negligible signal, PA1 that probing is possible in a homogeneous solution, PA1 that hybridization can be demonstrated rapidly, without delay, PA1 that the amount of NA can be quantified in real time, PA1 that particular NA sequences can be demonstrated in samples containing active enzymes, such as nucleases and proteases, PA1 that presence of a particular NA can be demonstrated in vivo, PA1 that the presence of a particular NA can be demonstrated with inexpensive equipment, PA1 that presence of an arbitrary sequence can be demonstrated selectively, PA1 that probing can be performed in a large temperature range, PA1 that people using the invention should not get exposed to hazardous chemicals, PA1 and that people using the probe should not require special training or particular experience.
To be able to utilize the entire potential of hybridization methods in diagnosis and research it is necessary to have a technique to detect hybridization in a solution using probes that by themselves generate low or negligible signals, but produce an observable response upon hybridization to target sequence. It is also desirable that the probe can be used in vivo without having a deleterious effect on tissue and cells. It should also allow real time detection. Of course, it should also be possible to use the probe for traditional hybridization. It is also desirable that the probe generates a signal that can be detected by the naked eye. The present invention fulfills these requirements to a reasonable degree.